WO2013050644A1 - Enantiomers of 2-hydroxy derivatives of fatty acids - Google Patents

Abstract

The invention relates to enantiomers of 2-hydroxy derivatives of fatty acids. The invention refers to the synthesis and purification of 2-hydroxy derivatives of fatty acids, and to the method for separating the enantiomers (or optical isomers) [-](S) and [+](R) of 2-hydroxy derivative compounds of fatty acids, to the actual enantiomers, to pharmaceutical compositions that include same and to the use thereof as drugs, as well as to in vitro diagnosis/prognosis methods and to methods for assessing the potential use of the molecules of the invention, in various diseases, as well as to the use thereof for regulating certain enzymes and to the study of the activity and effects thereof.

Description

 ENANTIÓMERO S DE 2-HIDROXIDERI FATTY ACID VADOS FIELD OF THE INVENTION

The present invention refers to a method of synthesis of racemic products and the separation of their optical isomers [-] (which corresponds to the S enantiomer) and [+] (which corresponds to the R enantiomer) of 2-hydroxy derivative compounds from fatty acids, to the isolated enantiomers themselves, to pharmaceutical compositions that comprise them, and to their use as medicines in the treatment of diseases whose common etiology is based on alterations (of any origin) of cell membrane lipids such as, for example : alterations in the level, in the composition or in the structure of said lipids, as well as in the treatment of diseases in which the regulation of the composition and membrane lipid structure induces the reversal of the pathological state. The therapeutic effect is preferably achieved through the regulation (activation or inhibition) of the activity of the enzyme ceramide: phosphocholine choline phosphotransferase (also known as sphingomyelin synthase or EC 2.7.8.27, IUBMB Enzyme Nomenclature), or the level of its product, sphingomyelin (SM). As a result of the activity of this enzyme and the production of MS and its accumulation in the membrane, in the tumor cell, there is an increase (up to 600% increase) of the glia acid fibrillary protein (PFAG) and a reduction (up to more than 90%) in the levels of the enzyme dihydrofolate reductase (DHFR). Both PFAG and DFIFR can therefore be used for the detection of the therapeutic efficacy of drugs for the treatment of diseases mediated by the activity of sphingomyelin synthase or SM itself. The present invention also refers to the development of a kit that is based on the detection of said diseases.

Additionally, both the enzyme EC 2.7.8.27, and sphingomyelin (SM), can be used as molecular markers of the effect of the compounds of the present invention on the diseases described above. Therefore, the present invention also encompasses an in vitro method for the diagnosis / prognosis of said diseases based on the evaluation of the regulation of the activity or level of said EC enzyme 2.7.8.27, and / or the level of MS, PFAG or DFIFR; as well as kits that include means specially designed to carry out said diagnoses / forecasts. These methods and kits would be based on the determination of the changes induced by treatments with the molecules mentioned in the present invention or in the possibility of changing the molecular entities indicated above with said treatments.

In addition, the enzyme EC 2.7.8.27 and / or SM can be used as therapeutic targets to which to direct molecules capable of reversing their altered state and, consequently, treating those pathological processes that had developed, or were to be developed in a future, as a result of the abnormal activity of the enzyme EC 2.7.8.27 or the inappropriate level of SM. Thus, both the enzyme EC 2.7.8.27, as well as the SM itself, can be the basis for the design of screening procedures for candidate compounds in order to achieve molecules that, such as enantiomers [-] (also called S) and [+] (corresponding to the R form) of 2-hydroxy derivative fatty acid compounds of the invention, have the ability to regulate the activity of the enzyme EC 2.7.8.27 and / or the level of SM, exerting a therapeutic effect. Thus, the present invention, due to its application spectrum, is likely to be encompassed in the field of medicine and pharmacy in general. It should be noted that since regulatory agencies in pharmaceutical matters require the existence of methods or kits to monitor the efficacy of a compound with a certain therapeutic activity, both the description of the compounds, their synthesis, their therapeutic scope and their detection should be considered parts of this invention.

STATE OF THE TECHNIQUE

Cell membranes are structures that define the entity of the cells and the organelles contained therein. In the membranes or in their vicinity most of the biological processes occur. Lipids not only have a structural role, but regulate the activity of important processes. Moreover, the regulation of the lipid composition of the membrane also influences the location or function of important proteins involved in the control of cellular physiology, such as G proteins or PKC (Escribá et al, 1995; Escribá et al, 1997; Yang et al, 2005; Martínez et al, 2005). These and other studies demonstrate the importance of lipids in the control of important cellular functions. In fact, numerous human diseases such as (among others): cancer, cardiovascular diseases, neurodegenerative processes, obesity, metabolic disorders, inflammation, infectious diseases and autoimmune diseases, have been linked to alterations in the levels or composition of lipids present in the Biological membranes, evidencing, in addition, the beneficial effects of treatments with other fatty acids than those of the present invention and regulating the composition and structure of membrane lipids, and can be used to reverse these diseases (Escribá et al, 2006).

The lipids that are ingested in the diet regulate the lipid composition of cell membranes (Alemany et al, 2007). Likewise, different physiological and pathological situations can change the lipids present in cell membranes (Buda et al, 1994; Escribá et al, 2006). Changes in the lipid composition of the membranes influence cell signaling, and may lead to the development of diseases or to reverting (Escribá et al, 2006). Different studies conducted in recent years indicate that membrane lipids play a much more important role than they had been assigned so far (Escribá et al, 2008). An example of this importance is the fish that live in rivers with variable temperature, whose lipids undergo significant changes (changes in abundance and types of membrane lipids) when the temperature drops from 20 ° C (summer) to 4 ° C (winter ) (Buda et al, 1994). These changes allow the maintenance of their functions in cell types of very diverse nature. Therefore, it could be said that membrane lipids can determine the good or malfunction of multiple cell signaling mechanisms. Since a diseased organism is so because its cells are diseased, alterations in membrane lipids can lead to diseases. Similarly, therapeutic, nutraceutical or cosmetic formulations, focused on regulating membrane lipid levels, can prevent and reverse (cure) pathological processes. In addition, numerous studies indicate that the consumption of saturated and trans-monounsaturated fats is related to the deterioration of health. Vascular and tumor diseases, among others, have been directly related to this type of lipid (Stendery and Dyerberg, 2004). The deterioration of an organism is manifested in the appearance of these and other types of diseases. Cell membranes constitute the selective barrier through which a cell exchanges metabolites and information with other cells and with the surrounding extracellular environment. However, membranes perform other very important functions at the cellular level. On the one hand, they support proteins involved in receiving or issuing messages that control important organic parameters. These messages, mediated by numerous hormones, neurotransmitters, cytokines, growth factors, etc., activate membrane proteins, which propagate the signal received into the cell through other proteins, some of which are also located in the membrane. Since (1) these systems function as amplification cascades and (2) that membrane lipids can regulate the location and function of said proteins, the lipid composition of the membranes can have an important impact on cellular functionality. Specifically, the interaction of certain proteins (called peripherals, such as G proteins, protein kinase C, Ras protein, etc.) with the cell membrane depends on its lipid composition (Vógler et al, 2004; Vógler et al, 2008). On the other hand, the lipid composition of cell membranes is influenced by the type and abundance of ingested lipids (Perona et al, 2007). From this it follows that lipid intake can regulate the lipid composition of membranes, which in turn can control the interaction (and therefore the activity) of important cellular signaling proteins (Yang et al, 2005).

The fact that membrane lipids can control cell signaling means that they can also regulate the physiological state of the cells. In fact, both negative and positive effects of lipids on health have been described (Escribá et al, 2006; Escribá et al, 2008). Preliminary studies have shown that 2-hydroxyoleic acid, which is a monounsaturated fatty acid, is capable of reversing certain pathological processes, such as overweight, hypertension or cancer (Alemany et al, 2004; Martínez et al, 2005; Vógler et al, 2008).

Cardiovascular diseases are frequently associated with hyperproliferation of the cells that constitute the cardiac and vascular tissues. This hyperproliferation of cardiovascular cells results in deposits in the internal lumen of the vessels and cavities of the cardiovascular system that result in a wide range of diseases, such as hypertension, atherosclerosis, ischemia, heart attacks, etc. (Schwartz et al, 1985). In fact, the development of drugs that prevent cell proliferation for the prevention and treatment of cardiovascular diseases has been suggested (Jackson et al, 1992).

Obesity or overweight is caused by an alteration between the balance of intake and energy expenditure that is due, in part, to alterations in the mechanisms that regulate these processes. On the other hand, this pathology is characterized by hyperplasia (increase in the number of cells) or hypertrophy (increase in size) of adipose tissue cells, adipocytes. Numerous studies show that fatty acids, either free or as part of other molecules, can influence a series of parameters related to energy homeostasis, such as body fat mass, lipid metabolism, thermogenesis or intake, among others ( Vógler et al, 2008). In this sense, the modification of fatty acids could be a strategy to regulate energy homeostasis and, therefore, body weight. In fact, in the present invention, it is shown how low levels of SM in cells are associated with increased cell proliferation and that said alteration is related to the pathological state of human cells. Moreover, the regulation of the enzyme EC 2.7.8.27 by means of the molecules described in the present invention are capable of normalizing the levels of MS in pathological cells and, therefore, reversing the pathophysiological alterations described herein. In the context of metabolic pathologies, in addition to obesity, lipid intake also determines the appearance of other pathological processes, such as hypercholesterolemia, hypertriglyceridemia, diabetes or metabolic syndrome (Sloan et al, 2008)

Neurodegenerative processes give rise to a series of diseases with different manifestations, but with the common characteristic of being caused by degeneration of the cells of the central and / or peripheral nervous system. Some of these neurodegenerative processes involve a significant reduction in the cognitive capacity of patients, such as Alzheimer's disease or senile dementia. Others lead to motor-type alterations, such as Parkinson's disease or different types of sclerosis. Finally, certain neurodegenerative diseases can lead to processes in which blindness, hearing problems, disorientation, mood alterations, etc. develop. An example of a well-characterized neurodegenerative disorder is Alzheimer's disease, in which the formation of senile plaques has been observed, composed of remains of membrane proteins (such as the β-amyloid peptide) erroneously processed, which accumulate outside of the cells, and clews of neurofilaments of Tau protein, which appear inside the cell. This process has been associated with alterations in cholesterol metabolism and the consequent alteration of cholesterol levels in membranes (Sagin et al, 2008). In fact, the development of this disease is related to other diseases in which alterations of lipid metabolism have been described, and more specifically of cholesterol, such as cardiovascular diseases.

On the other hand, sclerosis and other neurodegenerative processes are related to "demyelination", whose net result is the loss of lipids in the neuronal axon sheath, with the consequent alterations in the process of propagation of electrical signals that this implies. Myelin is a lipid layer that surrounds the axons of many neurons and is formed by a succession of spiral folds of the glia cell plasma membrane (Schwann cells). Therefore, it is clear that lipids play an important role in the development of neurodegenerative diseases. Moreover, it has been proven that unmodified natural polyunsaturated fatty acids have a moderate preventive effect on the development of neurodegenerative processes (Lañe et al, 2005).

Metabolic diseases form a set of pathologies characterized by the accumulation or deficit of certain molecules. A typical example is the accumulation of cholesterol and / or triglycerides above normal levels. The increase in cholesterol and / or triglyceride levels, both at the systemic level (for example the increase in plasma levels) and at the cellular level (for example in cell membranes) is associated with alterations in cell signaling that lead to dysfunctions at various levels, and that are normally due to errors in the activity of certain enzymes or the control of said proteins. Among the most important metabolites are hypercholesterolemia (high cholesterol levels) and hypertriglyceridemia (high triglyceride levels). These diseases have high incidence, morbidity and mortality rates, so their treatment is A necessity of the first order. Also, ingested lipids can determine the onset of diabetes (Sloan et al, 2008).

The protective role of certain unsaturated fatty acids on certain diseases has already been described by different researchers. In this way, unsaturated fatty acids slow the development of cancer and have positive effects against the development of cardiovascular diseases, neurodegenerative, metabolic diseases, obesity, inflammation, etc. (Trombetta et al, 2007; Jung et al, 2008; (Florent et al, 2006). However, the pharmacological activity of these compounds is very limited due to their rapid metabolization and short half-life in the blood. both the development of unsaturated fatty acids with a slower metabolization seems necessary, a consequence of which their presence in the cell membrane is increased, in comparison to the unsaturated fatty acids so far used, and that facilitate the interaction of peripheral proteins of cell signaling The molecules described in the present invention have the structural characteristics that determine a positive effect on the health of certain natural fatty acids, together with molecular modifications that enhance the effect of the original molecules and also prevent their rapid metabolization, both essential characteristics to determine its pharmacological activity.

Deepening the importance of cell membrane lipids, sphingolipids or sphingophospholipids are an important class of cell membrane lipids and are the most abundant in the tissues of more complex organisms. Sphingolipid molecules have unfriendly properties, that is, both hydrophobic and hydrophilic, which allows them to play an important role in the formation of biological membranes. Some of the glycosphingolipids are found on the surface of red blood cells and the rest of cells acting as antigens and constituting blood groups.

Therefore, sphingolipids are of great biological importance because of the role of cell signaling they play. Specifically, SM is a very abundant type of sphingolipid in the cell membranes of all organisms (Huitema et al, 2004). It is mainly located in the outer monolayer of the plasma membrane where it has an essential function in the formation of microdomains called lipid rafts, which are specialized areas of the cell membrane with important functions in cell signaling, since these domains concentrate proteins that interact with each other thanks to the approximation that derives from their binding to lipids (Simons and Toomre, 2000) . The enzyme EC 2.7.8.27 is responsible for the synthesis of MS by transferring a phosphocholine (from phosphatidylethanolamine or phosphatidylcholine) to the primary hydroxyl group of ceramide to form MS and 1,2-diacylglycerol (DAG). This enzyme occupies a central position in the metabolism of sphingolipids and glycerophospholipids. EC 2.7.8.27 is located in the plasma membrane, in the Golgi apparatus and its activity in the nuclear membrane and in chromatin has also been detected (Albi et al, 1999). EC 2.7.8.27 also acts as a regulator of ceramide and diacylglycerol (DAG) levels, both of which are molecules that regulate cell death programmed by apoptosis and autophagy (Jiang et al, 2011; Van Helvoort et al, 1994 ; Tafesse et al, 2006). The polar head of the SM is very bulky and prevents the anchoring of proteins, such as Ras, which have branched lipids (such as isoprenyl, farnesyl or geranylgeranyl moieties), while favoring the anchoring of other proteins that have saturated fatty acid residues ( as myristic or palmitic acids). This results in the inactivation of the MAP kinase signaling pathway, which is followed by a series of molecular events that include altering levels of PFAG and DHFR proteins. Therefore, given the importance of the enzyme EC 2.7.8.27 and the SM in the correct functioning and structure of the cell membrane, and the known relationship between both structural and functional alterations of the lipids located in the cell membrane with the triggering of several diseases such as cancer, cardiovascular disease, obesity, neurodegenerative and metabolic diseases; it would be very important to find compounds capable of regulating the activity of said enzyme and, consequently, the level of MS, and thereby being able to reverse pathologies whose origin is due to an abnormal enzyme activity and / or an altered level of MS. Since currently the drug marketing regulatory agencies, such as the Spanish Medicines Agency, the European Medicines Agency (EMA), the Food and Drug Administration, request the existence of effectiveness monitoring methodologies of medications, it is advisable to identify molecules whose Changes in expression or activity (among others) predict the efficacy of a compound to ensure that a patient receives appropriate medication. Therefore, those that allow the correct application of the treatments can be considered as related inventions. The present invention refers to the synthesis of a series of 2-hydroxy derivative compounds of fatty acids, the separation of their racemic forms and their therapeutic applications. In addition, the present invention also includes the description of the cell targets of their activity and, in addition, of biomarkers that allow determining the efficacy of said compounds, as well as the processes used for this. Additionally, it is also important to find compounds capable of regulating the activity of other enzymes involved in lipid metabolism, for example the enzyme serine palmitoyl transferase (EC 2.3.1.50); or the enzyme stearoyl-CoA desaturase (ECD, EC 1.14.19.1) responsible for the synthesis of oleic acid. Ceramide produced by the activity of EC 2.3.1.50 is a lipid molecule of great biological interest. An important role related to ceramide is its participation in the induction of apoptosis, also called programmed cell death (Lladó et al, 2010). Apoptosis is a very regulated biological process that serves to eliminate cells that are not useful or that compromise the health of the body. In this sense, it is common for tumor cells to develop molecular mechanisms to escape apoptosis (Lladó et al, 2010).

DESCRIPTION OF THE INVENTION Brief description of the invention

Preferably, the present invention focuses on resolving alterations in the cellular levels of SM, resulting in compounds capable of reversing the altered expression level of the enzyme EC 2.7.8.27 by activation (in case the enzyme is under-expressed or that has a reduced activity) or through its inhibition (in case the enzyme is overexpressed or has an increased activity), managing to control the levels of SM synthesized by said enzyme and, consequently, reverse the processes pathological due to enzyme deregulation or abnormal levels of MS. Thus, for the attainment of said objective, in the present invention a method of synthesis of molecules in their racemic form and subsequent isolation of the [-] and [+] enantiomers of 2-hydroxy derivative fatty acid compounds, was carried out. which, as demonstrated in the examples below, are capable of regulating the activity of the enzyme EC 2.7.8.27, and, consequently, the level of SM synthesized. These fatty acids have a longer half-life in the blood than natural fatty acids. In fact, the present invention refers to pharmaceutical compositions comprising said enantiomers, and their use as medicaments in the treatment of diseases whose common etiology is based on alterations (of any origin) of cell membrane lipids such as: alterations in the level, in the composition or in the structure of said lipids, as well as in the treatment of diseases in which the regulation of the composition and membrane lipid structure induces the reversal of the pathological state. The therapeutic effect is preferably achieved through the regulation (activation or inhibition) of the activity of the enzyme EC 2.7.8.27, and / or the level of its product, the SM, or even the level of PFAG and / or of DHFR.

In addition to the regulation made on the enzyme EC 2.7.8.27, the present invention demonstrates that, for example in U118 cells, compound 20HOA regulates the activity of other enzymes involved in lipid metabolism. Thus, the enzyme EC 2.3.1.50 was investigated. In the present invention, it is shown that 20HOA stimulates the activity of CD 2.3.1.50 (Example 8 and Figure 9), causing programmed cell death in human leukemia cells. On the other hand, an important reduction in oleic acid levels was also found in the membranes of U118 cells treated with 20HOA, demonstrating that 20HOA is a potent inhibitor of EC 1.14.19.1, an enzyme responsible for the synthesis of oleic acid from of stearic acid (Example 9 and Figure 10). In this sense, it should be noted that both EC 2.7.8.27 and EC 2.3.1.50 are enzymes corresponding to the metabolism of sphingolipids, which are related to belonging to the metabolic pathway of the same type of molecules. On the other hand, the enzyme EC 1.14.19.1 is a fatty acid modifier. The relationship with the other two enzymes is that sphingolipids always carry fatty acid chains in their structure. Since each fatty acid has different properties than sphingolipids, the fact that they can be modified affects their biological activity. Thus, it can be affirmed that the three enzymes mentioned in the present invention are closely related and therefore it is normal that the same molecule can regulate the activity of the 3 enzymes.

The enantiomers [-] (also S isomer) and [+] (also R isomer) of the invention differ in the direction of deflection of polarized light. If the optical isomer deflects the polarized light to the right (in orientation with the hands of the clock) it is represented by the sign [+] (it is the dextrogiro isomer or dextrous form). On the other hand, if the optical isomer deflects the polarized light to the left (in a counter-clockwise direction), it is represented by the sign [-] (it is the levographic isomer or levo form).

Specifically, the present invention evidences that the [-] enantiomer of 2-hydroxy derivative compounds of fatty acids acts as activator of the enzyme EC 2.7.8.27 by positively regulating the synthesis of SM, a sphingolipid that, as explained above, is mostly in the membranes of human and animal cells, and indispensable for the correct structuring of the lipid bilayer and functioning of the cell. Therefore, said enantiomer [-] can be used for the preparation of a pharmaceutical composition for the treatment of those pathologies whose common etiology is structural and / or functional alterations of lipids located in the cell membrane, such as: cancer, obesity , hypertension, hypertriglyceridemia, hypercholesterolemia or diabetes, etc., due to an abnormally low activity of said enzyme EC 2.7.8.27. Likewise, the racemic form is activating this enzyme, since the activity of the [-] isomer predominates, which is the active one, over the [+] isomer, which does not induce the activity of the enzyme. In this sense, the positive activity of the racemic form is considered to be due to the fact that for the activity of this enzyme an activation that induces the synthesis of new molecules of SM is more important, than the inhibition that can silence enzyme molecules than previously They were not active. In any case, the activating power of the racemic form is inferior to that of the enantiomer [-], which explains its lower therapeutic activity.

As shown in the examples of the invention, it is important to note that the [-] enantiomer of 2-hydroxy derivative fatty acid compounds exhibits an improved therapeutic effect with respect to the racemic (which contains equal amounts of the two enantiomers). In addition, it is also noteworthy that the [-] enantiomer of 2-hydroxy derivative fatty acid compounds has less toxicity and side effects than the racemic one and that the [+] enantiomer (see Table 3, Example 7).

In addition, the present invention also demonstrates that the [+] enantiomer of 2-hydroxy derivative fatty acid compounds acts as an inhibitor of said EC enzyme 2.7.8.27 by negatively regulating the synthesis of SM, and can be used in basic research for the study of regulation of the EC enzyme 2.7.8.27 itself, or for the treatment of diseases characterized by an abnormally high activity of the enzyme EC 2.7.8.27, and / or an abnormally high level of SM, such as cystic fibrosis (Slomiany et al, 1982). On the other hand, high cholesterol and triglyceride levels have been associated with significant cardiovascular disorders. In this sense, cholesterol is usually associated with MS to form very ordered membrane domains, referred to in the scientific literature lipid rafts or Lo (liquid ordered, that is, ordered liquid). The increase in cholesterol favors the increase of these lipid regions, which implies changes in cell signaling that can lead to different diseases or cardiovascular and metabolic alterations. Therefore, reducing the levels of MS in cases of hypercholesterolemia and hypertriglyceridemia can help lower cholesterol and triglyceride levels in plasma and membranes. In this way, the enantiomer [+] would have a protective role in certain types of disorders, such as hypercholesterolemia and hypertriglyceridemia, since it induces reductions in serum levels of these lipids and reductions in MS levels that would concur in a reduction of The density of lipid rafts in the cells.

Additionally, since the enzyme EC 2.7.8.27 is responsible for the synthesis of SM and, therefore, for the correct structure and function of the cell membrane, both the enzyme and the SM itself could be considered as molecular markers, usable to lead to carry out a method of diagnosis and / or prognosis in vitro of diseases based on alterations in the cell membrane. Such changes do not occur naturally, but are caused by the activity of the compounds described in the present invention. In addition, it has been found that the compounds of the present invention also regulate the levels of DHFR and PFAG proteins. Consequently, the present invention also refers to a method / kit for Carry out the diagnosis or prognosis of pathologies associated with altered levels with respect to the normal DHFR and PFAG proteins. Said kit comprises reagents or means capable of evaluating the activity of the enzyme EC2.7.8.27, and / or the level of SM, DFIFR or PFAG and, consequently, implementing said diagnosis / prognosis as a method to follow the efficacy of the treatment of patients

For the same reason, said enzyme EC2.7.8.27 and / or its product, SM, can be considered as therapeutic targets to which to direct molecules capable of regulating the activity of the enzyme, and / or the level of MS, and , consequently, to reverse those pathological processes that would have developed, or were going to develop in the future, as a result of the alteration in the activity of the enzyme or in the level of SM, DFIFR or PFAG. Thus, by way of example, the [-] enantiomer of the invention serves to illustrate the possible use of the enzyme EC 2.7.8.27 as a therapeutic target, by activating its enzymatic function in pathological processes in which said function is deficient, being able to restore the level of SM at normal levels. On the other hand, the measurement of the activity of the enzyme EC 2.7.8.27, and / or the levels of SM, and / or the levels of PFAG, and / or the levels of DHFR, would also be useful for performing selection procedures ( screening, in English) of candidate compounds with the aim of achieving other molecules that, such as the [-] and [+] enantiomers of the invention, had the ability to regulate the activity of the enzyme EC 2.7.8.27, and / or the level of SM, and / or the level of PFAG, and / or the level of DHFR, being able to reverse pathological processes.

Consequently, the present invention demonstrates the particular importance of selecting compounds with exclusive structural characteristics such as: fatty acids with at least one double bond, with a total of carbon atoms (C) equal to or less than 20, and a substituted carbon, particularly with a hydroxyl radical (OH), on carbon 2 (or carbon a).

Specifically, the compounds referred to in the present invention are the [-] and [+] enantiomers of Formula I:

HOOC-HOCH- (CH ₂ ) „- (CH = CH-CH2) _é - (CH ₂ ) _c -CH3

(I) where a, b and c can take independent values between O and or, taking into account that the total carbon sum of the molecule is <20.

Furthermore, it is established that the preferred therapeutic form of the present invention is the [-] enantiomer (corresponding to the steric configuration S) of Formula I, which is presented as the most effective way in the activation of the enzyme EC 2.7. 8.27, ahead of the racemic form, and the [+] enantiomer (corresponding to the steric configuration R) of Formula I which is presented as an inhibitor of the enzyme EC 2.7.8.27.

In a preferred aspect, the present invention makes special reference to the [+] and [-] enantiomers of Formula I with the following values of a, b and c:

Table 1

In a particularly preferred aspect the present invention refers to the [-] enantiomer of the formula [-] HOOC-HOCH- (CH ₂ ) _é - (CH = CH-CH ₂ ) i- (CH ₂ ) _(í -CH _3; named, for the purposes of the present invention, [-J20HOA.

In another particularly preferred aspect the present invention refers to the [+] enantiomer of the formula [+] HOOC-HOCH- (CH ₂ ) _é - (CH = CH-CH ₂ ) i- (CH ₂ ) _(í -CH _3; named, for the purposes of the present invention, [+ J20HOA.

As an example, diseases characterized by a deficit in the activity of the enzyme EC 2.7.8.27, and, consequently, by an abnormally low level of SM in cell membranes, and that could be treated or prevented with the enantiomer [- ] of the invention are:

• Cancer: prostate cancer, breast cancer, pancreatic cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer (see Table 2). Vascular pathologies: hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia, etc.

 Metabolic pathologies: diabetes, metabolic syndrome or obesity.

 Other pathologies: spinal cord injury, Alzheimer's disease, stroke, sclerosis, cellulite, etc.

Therefore, more specifically, the first aspect of the present invention refers to an [-] or [+] enantiomer of a compound of Formula I and / or at least one of its pharmaceutically acceptable salts

HOOC-HOCH- (CH ₂ ) „- (CH = CH-CH2) _é - (CH ₂ ) _c -CH3

 (i) characterized in that a, b and c can take independent values between O and or, provided that the total number of carbons is <20. In a preferred aspect of the invention the compound results from the selection of at least one of the following combinations of values a, b and c: a = 6, b = \ and c = 6; a = 6, b = \ and c = 4; a = 6, b = 2 and c = 3; a = 6, b = 3 and c = 0; and a = 3, b = 3 and c = 3.

In addition, a preferred aspect of the present invention refers to the compounds of the formula: [+] HOOC-HOCH- (CH ₂ CH = CH H ₂ ) / - (CH ₂ ) 6-CH ₃ By way of example, diseases characterized by an excess in the activity of the enzyme EC 2.7.8.27, and, consequently, by an abnormally high level of SM in cell membranes, and which could be treated or prevented with the enantiomer [+] of the invention are fibrosis cystic, hypercholesterolemia and hypertriglyceridemia.

Another particularly preferred aspect of the present invention refers to the compounds of the formula: [-] HOOC-HOCH- (CH2) ₆ - (CH = CH-CH2) 7- (CH ₂ ) _í; -CH3.

A second aspect of the present invention refers to the use of at least one compound of the aforementioned for the preparation of a pharmaceutical composition for the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane , due to deregulation of the activity of the enzyme EC 2.7.8.27, of the level or concentration of SM, of the level of PFAG or of the level of DHFR, in cells in general and in membranes, in particular.

In addition to the present invention, it covers at least one compound of the aforementioned for use in the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane, due to the deregulation of the activity of the EC enzyme 2.7.8.27, of the level or concentration of SM, of the level of PFAG or of the level of DHFR, in cells in general and in membranes, in particular.

Even the present invention refers to the pharmaceutical composition itself comprising at least one of said compounds and, optionally, pharmaceutically acceptable carriers. Thus, the compounds of the present invention can be administered independently or formulated in pharmaceutical compositions where they are combined with excipients such as: binders, fillers, disintegrators, lubricants, coaters, sweeteners, flavorings, colorants, transporters, etc., and combinations thereof. Also, the compounds of the invention can be part of pharmaceutical compositions in combination with other active ingredients.

For the use of the compounds of the invention as medicaments, their administration can be carried out by any route such as, for example, enterally (through the digestive system), orally (by pills, tablets or syrups), rectally (by suppositories or enemas), topically (through creams or patches), inhalation, parenterally injected, intravenous injection, intramuscular injection or subcutaneous injection, as indicated above or in any type of pharmaceutically acceptable form, such as : methyl, ethyl, phosphates, other radicals of the ester, ether, alkyl type, etc.

In a preferred aspect, the invention relates to the use of the compound [-] HOOC-HOCH- (CH2) - (CH = CH-CH2) ₇ - (CH2) ¿-CH3 for the preparation of a pharmaceutical composition intended for treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations in the cell membrane due to a deficit in the activity of the enzyme EC 2.7.8.27, an abnormally low level of MS in the cell membrane, an abnormally low level of PFAG or an abnormally high level of DHFR; pathologies being preferably selected from: preferably prostate cancer, breast cancer, pancreatic cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer; vascular pathologies preferably hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia or metabolic pathologies; Metabolic pathologies preferably: diabetes, metabolic syndrome or obesity.

The present invention also refers to the compound [-] HOOC-HOCH- (CH2) 6- (CH = CH-CH2) _/ - (CH2) 6-CH ₃ to be used in the treatment and / or prevention of pathologies whose etiology common are structural and / or functional alterations in the cell membrane due to a deficit in the activity of the enzyme EC 2.7.8.27, an abnormally low level of SM in the cell membrane, an abnormally low level of PFAG or an abnormally high level of DHFR; pathologies being preferably selected from: preferably prostate cancer, breast cancer, pancreatic cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer; vascular pathologies preferably hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia or metabolic pathologies; Metabolic pathologies preferably: diabetes, metabolic syndrome or obesity.

In another preferred aspect, the invention refers to the use of the compound of formula [+] HOOC-HOCH- (CH2) ₆ - (CH = CH-CH2) ₇ - (CH2) ₆ -CH3 for the preparation of a pharmaceutical composition intended to the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations in the cell membrane due to an excess in the activity of the enzyme EC 2.7.8.27, an abnormally high level of SM in the cell membrane, a level abnormally high PFAG or an abnormally low level of DHFR; being the pathology, for example, cystic fibrosis hypercholesterolemia and triglyceridemia.

The invention also refers to the compound of the formula [+] HOOC-HOCH- (CH2) _Í ;-( CH = CH-CH2) 7- (CH2) _Í ; -CH ₃ to be used in the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations in the cell membrane due to an excess in the activity of the enzyme EC 2.7.8.27, an abnormally high level of SM in the cell membrane, an abnormally high level of PFAG or an abnormally low level of DHFR; being the pathology, for example, cystic fibrosis hypercholesterolemia and triglyceridemia. A third aspect of the present invention refers to a method of treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane, due to deregulation of the activity of the enzyme EC 2.7.8.27, the SM level, the PFAG level or the DHFR level; which comprises the administration to the patient of a therapeutically effective amount of at least one compound of the aforementioned or compositions comprising them.

In a preferred aspect, the invention refers to a method for the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations in the cell membrane due to a deficit in the activity of the enzyme EC 2.7.8.27 and / or at an abnormally low level of SM in the cell membrane, which comprises administering to the patient a therapeutically effective amount of the compound [-] HOOC-HOCH- (CH2) ₆ - (CH = CH-CH2) ₇ - (CH2) _í; -CH3 or a composition that includes it. In a preferred aspect, the pathologies are preferably selected from: preferably prostate cancer, breast cancer, pancreas cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer; vascular pathologies preferably hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia or metabolic pathologies; Metabolic pathologies preferably: diabetes, metabolic syndrome or obesity. In another preferred aspect, the invention refers to a method of treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations in the cell membrane due to an excess in the activity of the enzyme EC 2.7.8.27 and / or at an abnormally high level of SM in the cell membrane, which comprises the administration of a therapeutically effective amount of the compound [+] HOOC-HOCH- (CH2) ₆ - (CH = CH-CH2) ₇ - (CH2) ₆ -CH3 or of a composition that understand In a preferred embodiment the pathology is, for example, cystic fibrosis, hypercholesterolemia and hypertriglyceridemia.

For the purposes of the present invention, "therapeutically effective amount" is understood as that which reverts the disease or prevents it without showing adverse side effects. "Therapeutically effective amount" would also be understood as that which, producing a significant therapeutic effect, had an acceptable level of toxicity when the treated disease was very serious or fatal.

A fourth aspect of the present invention refers to an in vitro method for the selection of candidate compounds useful in the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane, which includes the evaluation of changes produced on the activity of the enzyme EC 2.7.8.27, the level of SM, the level of PFAG or the level of DHFR in the presence of said candidate compound. That is, the present invention comprises the use of the enzyme EC 2.7.8.27, of SM, PFAG or DHFR as therapeutic targets, with the purpose of designing therapeutic tools capable of altering their activity (eg, Figure 5) or levels and to which to direct compounds with the aim of preventing and / or treating the pathologies described above.

The fifth aspect of the present invention refers to in vitro methods for the prognostic / diagnosis of pathologies whose common etiology is structural and / or functional alterations of the lipids located in the cell membrane comprising the determination of the deregulation of the activity of the enzyme EC 2.7.8.27 and / or the presence of an abnormal level of SM, PFAG or DFIFR in the cell membrane or other cell compartments. That is, the present invention comprises the use of the enzyme EC 2.7.8.27, or of SM, PFAG or DFIFR, as molecular markers through which to make the diagnosis and / or prognosis of the previously described pathologies. The importance of MS in the structure and activity of the cell membrane, and of the enzyme EC 2.7.8.27 as responsible for producing this phospholipid, make the detection of the concentration / cellular activity of both in tissues, plasma, cerebrospinal fluid, urine and / or other body fluids, can be used in the preparation of kits for detection, diagnosis and monitoring of different pathologies included in the present invention, such as, for example, cancer, hypertension, Obesity and metabolic diseases. Therefore, both SM and the enzyme EC 2.7.8.27, DHFR and PFAG constitute biomarkers for the detection of human diseases, and, as mentioned above, are therapeutic targets for the design of new therapies for humans. In a preferred aspect, the present invention refers to an in vitro method for the prone diagnosis / diagnosis of pathologies whose common etiology is structural and / or functional alterations of the lipids located in the cell membrane comprising the determination of a deficit in the EC enzyme activity 2.7.8.27, the presence of an abnormally low level of SM in the cell membrane, an abnormally low cellular level of PFAG or an abnormally high cellular level of DFIFR, where, preferably, the pathologies They are selected from: preferably prostate cancer, breast cancer, pancreas cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer; vascular pathologies preferably hypertension, arteriosclerosis, cardiomyopathies, stroke, angiogenesis, cardiac hyperplasia or metabolic pathologies; Metabolic pathologies preferably: diabetes, metabolic syndrome or obesity.

In another preferred aspect, the present invention refers to an in vitro method for the prone diagnosis / diagnosis of pathologies whose common etiology is structural and / or functional alterations of the lipids located in the cell membrane comprising the determination of an excess in the EC enzyme activity 2.7.8.27, the presence of an abnormally high level of SM in the cell membrane, an abnormally high level of PFAG or an abnormally low level of DHFR, where, preferably, the pathology is fibrosis cystic hypercholesterolemia and hypertriglyceridemia. The sixth aspect of the present invention refers to kits to be used in the prognostic / diagnostic method defined above, which comprises useful means for determining the activity of the enzyme EC 2.7.8.27, of the level of SM in the membrane cellular, DHFR levels / activity and / or GAPF levels. Such useful means comprise the TLC and HPTLC technique, gas chromatography, image analysis, absorption or fluorescence spectroscopy, optical microscopy, fluorescence, confocal microscopy, immunoblotting, immunocytochemistry, ELISA or similar techniques (RIA, dot / slot blot, EIA, etc.).

In a preferred aspect the kit is characterized in that the prognosis / diagnosis is carried out through direct quantification of sphingomyelin levels, and / or indirect quantification thereof through its precursors (for example phosphatidylethanolamine, phosphatidylcholine , ceramide, etc.) or its derivatives (for example sphingolipids). Preferably, the precursor is ceramide, the derivative is BD-Cer or BD-SM and indirect measurement is made through lisenin.

Another aspect of the present invention refers to a method of synthesis of racemic compounds and the isolation and purification of the enantiomers of the molecules of the invention comprising the following steps:

1. Hydroxylation in acidic medium of unsaturated fatty acid to produce the racemic product.

 2. Purification by recrystallization and precipitation of racemic fatty 2-hydroxy acid in its sodium salt form.

 3. Esterification of the racemic mixture of 2-hydroxy fatty acid in its sodium salt form with a solution of sulfuric acid in ethanol, preferably 10%.

 4. Enantioselective hydrolysis of the ester, catalyzed by pseudomonas fluorescens lipase (Amane Lipase, cas # 9001-62-1) at 25 ° C.

 5. Control of the progress of the reaction by liquid chromatography.

 6. Hydrolysis of the mixture with diluted HC1 to pH less than 2.

 7. Extraction of the products in methyl-terbutyl ether (MTBE) and washing of the organic phase.

 8. Removal of the solvent in vacuo to obtain a crude with the mixture of acid and ester.

 9. Separation of acid and ester by crystallization of the first.

10. Return to step 1) in the case of acid and step 2) in the case of the ester to start reprocessing each of the isolated fractions (acid and ester) until the desired enantiomeric purity is obtained (95% enantiomeric excess, which is equivalent to 97.5% of the desired enantiomer and 2.5% of the unwanted one). The separation and isolation of the two enantiomers of 20HOA is not known to date. Especially the isolation of the enantiomer [-] had not been possible to date due to its technical difficulty. The present invention also refers to an in vitro method of monitoring the cellular alterations produced on the diseased cells by the effect of the compounds of the present invention, or other compounds that act on the same process or on related cellular processes, producing the healing or improvement of the affected cells in the patient. That is, the present invention comprises the use of the enzyme EC 2.7.8.27, or of SM, DHFR or GAPF as molecular markers whose changes induced by the treatment with the molecules of the present invention allow to know if the patient responds to the treatment and, therefore, determine its effectiveness and the time that must be maintained. These methods can also make it possible to predict whether a patient has the possibility of responding to the treatment with the molecules of the present invention, so they can be used to make the diagnosis and / or prognosis of the previously described pathologies. Since the alteration of the membrane composition, and in particular of the levels of SM, results in changes in the levels of DHFR and PFAG proteins, a preferred aspect of the present invention refers to a method that allows to follow the changes induced by the molecules of the present invention or other molecules that perform a similar effect on pathological cells. Therefore, the use of MS, the enzyme EC 2.7.8.27, DHFR and / or PFAG offers the possibility that the applied therapy can change its levels or activity. The purpose of this determination is (1) the prediction of the efficacy of the treatment to avoid potentially non-responding patients following an ineffective therapy and (2) knowing the evolution of the patient to confirm that he responds to the therapy and know the doses to be delivered depending on the phase of the treatment, as well as the duration of these phases and the treatment itself as a whole. As these aspects are critical to avoid unnecessary pharmaceutical expenditure and to be able to apply the therapy in the most rational way possible, the large regulatory agencies in pharmaceutical matters request the existence of biomarkers and diagnostic systems as described here. Likewise, the present invention refers to a method for the treatment of patients suffering from the pathologies described above, which comprises the determination of the presence of said deregulation in the enzyme EC 2.7.8.27, of the level of MS, of the level of PFAG or DHFR level, and the treatment of the patient presenting said deregulation with the compounds of the invention.

Furthermore, the present invention refers to a method for selecting therapy for a patient with a pathology described in the present invention which comprises determining the presence of said deregulation in the enzyme EC 2.7.8.27, of the level of MS, of the level of PFAG or DFIFR level and the selection, based on said determination, of a therapy based on the compounds of the present invention.

For the purpose of illustrating the present invention, the following definitions are included:

• High / low activity of the enzyme EC 2.7.8.27: Enzymatic activity that results in the appearance of high or low levels, respectively, of SM in membranes. · High / low level of sphingomyelin: Low levels of sphingomyelin are considered to be less than 15% of total membrane phospholipids. High levels of sphingomyelin are considered to be 15% or more of this phospholipid, with respect to total phospholipids.

• High / low level of PFAG: A high level of PFAG is considered to be double or more (> 200%) of the normal levels present in glia cells, in reference to mg of total protein. A low level of PFAG is considered to imply the presence of levels of half or less (<50%) with respect to the normal levels present in glia cells, in reference to mg of total protein. · High / low level of DHFR: A high level of DHFR is considered to be double or more (> 200%) of the normal levels present in quiescent cells of any type, in reference to mg of total protein. A low level of DHFR is considered to imply the presence of levels of half or less (<50%) with respect to the normal levels present in quiescent cells of any type, in reference to mg of total protein. Brief description of the figures Figure 1.

A. The compound 20HOA induces a significant increase in the synthesis of MS in different human brain cancer cell lines (U118, SF767 and 1321N1), human lung cancer cells (A549) and human leukemia cells (Jurkat), but not in non-tumor cells (human lung fibroblast cells MRC5). This increase is, therefore, induced and specific for tumor cells. Likewise, the antitumor effect on these and other tumor lines has also been studied (see Table 2). The cells were treated vehicle (water with 5% ethanol, Control) or with the compound 20HOA, for 24 hours at a concentration of 200 μΜ. The ordinate axis values represent the mean ± ESM (standard error of the average of a number of experiments n = 3-5) of the levels of SM (percentage of total phospholipids), determined by chromatographic processes (TLC or HP-TLC , followed by spectroscopic determination and quantification by image analysis), as a percentage of total lipids compared to untreated (control) cell levels. The levels of MS measured in tumor cells are significantly lower than those of normal cells and the 20HOA compound only induced significant changes in cancer cells. These results indicate that the enzyme EC 2.7.8.27 is a therapeutic target for the treatment of cancer and a biomarker for the monitoring of the pathology and its evolution during the applied therapies, and that, on the other hand, the compound 20HOA is an activator of this enzyme and reverses the low levels of SM presented by tumor cells. In addition to the techniques described above to measure enzyme activity and MS levels, other techniques have been tested that have yielded very similar results, such as gas chromatography, confocal microscopy, fluorescence spectroscopy, etc.

B. Images of confocal microscopy showing the mapping of the cellular SM by lysinein binding, followed by the marking with a fluorescently labeled antibody, in U118 human glioma cells treated with vehicle (Control) or with 20HOA (200 μΜ). It can be seen in the tumor cells that the amount of SM in the membrane is low and that 20HOA treatments induce a dramatic increase in the amount of SM in the membrane, a clear indicator of the activation of EC enzyme 2.7.8.27. This experiment also shows clearly that the increase in the level of cellular SM is mainly due to an accumulation in the plasma membrane of the tumor cells.

C. Upper panels: Specific marking, by immunocytochemical techniques, of SM (left: lysenin followed by antibody labeled with fluorophore Alexa 488), of nuclear DNA with Hoechst 33258 (center: Hoechst), or both (right, merge). The figures correspond to sections of human lung cancer tumors (A549 cells) formed in immunosuppressed mice treated with vehicle (Control) or with [- J20HOA at 600 mg / kg -day or 900 mg / kg -day (oral, 50 days ). These results clearly show how MS levels increase significantly after induction of treatment with [-J20HOA, but they do not increase naturally in such tumors.

Central panels: Detail of the SM mapping (left), nuclear DNA (center), or both (right) in a cell present in a tumor cut developed in a mouse infected with human lung cancer (A549) and treated with [ -J20HOA (600 mg / kg -day, po, 50 days). The image shows the loss of nuclear DNA, a clear indicator of the onset of cell death, in parallel to the increase in MS.

Lower panel: The ordinate axis represents the quantification of the fluorescence intensity (arbitrary units) by confocal microscopy in sections of the tumors shown in the upper panel (mean ± standard error of the mean), from vehicle treated animals (Control ), or 20HOA at 600 mg / kg -day (T600) or 900 mg / kg · day (T900) for 50 days (po). *** P <0.001.

D. Determination of MS levels by fluorescence spectroscopy. Model membranes (liposomes) with different SM content were incubated in the presence of lisenin, first, and an anti-lysenin antibody labeled with fluorophore Alexa 488, then. The membrane bound fluorescence (SM levels) was separated from the unbound by centrifugation at 80,000 xg for 30 minutes. The precipitate was resuspended in a medium with detergent (1% sodium dodecyl sulfate) and the fluorescence (arbitrary units in the precipitate) in each pellet (ordered) was quantified according to the concentration of SM (bees). Any fluorophore that binds to the antibody or lysenin can be used for the detection of this lipid. Figure 2. Compound 20HOA induces an increase in the synthesis of MS in a manner dependent on its concentration and the time of treatment. The ordinate axis values represent the mean ± ESM of the levels of MS (percentage of total phospholipids) with respect to the control without treatment (n = 3-5). The levels were SM were determined by thin layer chromatography (HP-TLC, high performance thin layer chromatography), followed by image analysis or gas chromatography:

A: Ul 18 human glioma cancer cells treated at a concentration of 200 μΜ of 20HOA at different times (2, 6, 12, 24, 48 and 72 hours). The white bars correspond to cells treated with vehicle (control). B: U118 cells treated for 24 hours at different concentrations of 20HOA (25-400 μΜ).

Figure 3. The 20HOA compound induces an increase in nuclear SM. U118 cancer cells were treated for 24 hours with vehicle (control) or with 20HOA at a concentration of 200 μΜ. The ordinate axis values represent the mean ± ESM (n = 3-5) of the levels of SM with respect to the control (100%) without treatment.

Figure 4. The compound 20HOA acts directly on the enzyme EC 2.7.8.27. In A, B and C, in all cases, the cells or cell extracts or incubation medium were incubated with the fluorescent substrate of this BD-Cer enzyme (nitrobenzoxadiazol-yl) ceramide], in the presence or absence (control, white bars) of 20HOA (black bars). The lipids were then extracted and the levels of BD-SM formation determined by HPTLC (High performance Liquid Chromatography). The values are presented in the ordinate axis as the mean ± ESM (n = 3-5):

A: In vivo experiment of the activity of the enzyme EC 2.7.8.27: Ul 18 cells were incubated with NBD-Cer for 4 hours and then treated with compound 20HOA (200 μΜ) at different times (5, 15, 60 minutes and 24 hours). The cells were then homogenized and the metabolization of the NBD-Cer fluorophore was measured to become NBD-SM.

B: In vitro experiment of the activity of the enzyme EC 2.7.8.27 in cell extracts, in which the activity of the enzyme was measured after killing the cells. This experiment demonstrates that compound 20HOA is capable of activating the enzyme EC 2.7.8.27 by direct interaction. For these experiments, cell homogenates were incubated with compound 20HOA (200 μΜ) and BD-Cer for 2 hours.

C: Effect of compound 20HOA on the activity of isoforms 1 (cells) and 2 (medium) of the enzyme EC 2.7.8.27. The activation of the EC 2.7.8.27 (1) and EC 2.7.8.27 (2) isoforms was determined by testing their activity in the entire cell or on the cell surface, respectively.

Figure 5. Structure-function relationship in the activation of the enzyme EC 2.7.8.27. Increase of SM in U118 cells induced by different fatty acids (200 μΜ, 24 h). Control, vehicle; 18: ln-9 (octadecenoic acid); 2Me-18: ln-9 (2-methyloctadecenoic acid); 20H16: 1 (2-hydroxyhexadecenoic acid); 2OH-18: 0 (2-hydroxyoctadecanoic acid); 20HOA (20H-18: ln-9, 2-hydroxyoctadecenoic acid); 20H- 18: 2n-6 (2-hydroxylinoleic acid); 20H-18: 3n-6 (2-hydroxy-y-linolenic acid); 20H- 18: 3n-3 (2-hydroxy-a-linolenic acid); 2OH-20: 4n-6 (2-hydroxyarachidonic acid); 2OH-20: 5n-3 (2-hydroxyeicosapentaenoic acid); 20H-22: 6n-3 (2-hydroxyacosahexaenoic acid). The ordinate axis values represent the mean ± ESM (n = 3-5) of the SM levels, determined by thin layer chromatography, with respect to the control without treatment (100%). Fatty acids of 20 C atoms or more do not produce significant changes in the activity of this enzyme. The presence of other radicals in carbon 2 (such as H or CH ₃ ) and the lack of double bonds in the fatty acid structure resulted in inactive molecules

Figure 6

A: Changes in cell membrane composition are shown to induce the translocation of Ras protein from the membrane to the cytoplasm. Elevated membrane levels of compound 20HOA and SM prevent anchoring of the Ras protein in the membrane. The figure shows phase contrast (Ph.C) optical microscopy images of human glioma cells (SF767), and confocal microscopy, using a specific antibody against fluorescently labeled Ras, after 10 minutes (confocal 1) and 24 hours (confocal 2) treatment with vehicle (control) or compound 20HOA. B: The translocation of the Ras protein prevents the inactivation of the Raf protein, as well as the next activation in the signaling cascade of the MEK protein, as observed by the reduction in state of the phosphorylated (active) form of both proteins, determined by immunoblot in U118 cells grown in absence (Control) no presence of 150, 200 or 250 μΜ of 20HOA.

C: The previous molecular / cellular events lead to a dramatic reduction in the activity status of the MAP kinase protein (ERK1 and ERK2), also determined by immunoblotting with specific antibodies, at the concentrations of 20HOA indicated in B. D: Activity states (phosphorylated form levels) of Akt and EGFR also decrease after incubation with 250 μΜ of 20HOA.

In panels B to D, the ordinate axis indicates the percentage of phosphoprotein with respect to the untreated (control) SF767 cells. The abscissa axis indicates the concentration (micromolar) of compound 20HOA. E: Upper panel: DHFR expression levels in tumors derived from human glioma cells (SF767) implanted in nude mice that were treated with 20HOA for 50 days (600 mg / kg · day, po). The graph on the left shows the mean values (n = 4) of fluorescence determined by immunocytochemistry using a specific fluorescently labeled antibody and the photographs on the right correspond to a representative image. Lower panel: Expression of DHFR in human lung cancer cells (A549). All the experimental conditions and details of this graph are similar to the upper panel. These results demonstrate that DHFR levels vary in response to treatment with the molecules of the invention, so that the activity or levels of this protein can be used as biomarkers to track the therapeutic efficacy of the compounds described herein or compounds acting as similar form. It also follows that tumors in which DHFR levels are high may respond well to treatment with the compounds of the present invention, so this protein is a good biomarker to predict the possible efficacy of enantiomers of fatty acid derivatives. and subsequently demonstrate the efficacy of treatment with these compounds. F: Left panel: PAFG levels in serum of animals with human tumors (glioma) determined by immunoblotting. Naked mice were infected with SF767 cells and treated with vehicle (Glioma) or with 20HOA (T, 600 mg / kg · day, po, 28 days). Immunoreactivity against the fragmented peptide of the PFAG (see photo) in the serum of these animals, expressed as a percentage, was compared with that found in tumor-free and untreated animals (Control). Right panel: Idem, but determined by serum ELISA of animals treated for 7 days (7d600) or 28 days (28d600) with 600 mg / kg of 20HOA (po). A specific antibody against PFAG was used for all experiments. These results demonstrate that PFAG levels vary in response to treatment with the molecules of the invention, whereby the levels of this protein can be used as biomarkers to follow the therapeutic efficacy of the compounds described herein or compounds that act similarly. . It also follows that tumors in which PFAG levels are low may respond well to treatment with the compounds of the present invention, so this protein is a good biomarker to predict the possible efficacy of enantiomers of fatty acid derivatives. and subsequently demonstrate the efficacy of treatment with these compounds. Various techniques have been used to measure DHFR and PFAG, including electrophoresis, immunoblot, ELISA and the like, RT-PCR, etc. All these and similar techniques can be used to determine the levels or activity of DHFR and PFAG.

Figure 7. A) Specificity of the action of the [-] and [+] enantiomers, and of the +/- racemic mixture of 20HOA, in the activity of the enzyme EC 2.7.8.27. The figure shows the levels of SM (percentage of SM over total lipids) in U118 glioma cells after treatment with the racemic mixture of 2-hydroxyoleic acid (+/-), with compound [-] 20HOA and with compound [+] 20HOA. The cells were treated 24 hours at a concentration of 50 μΜ (50) and 100 μΜ (100). The ordinate axis values represent the mean ± ESM (n = 3-5) of the levels of SM determined by thin layer chromatography as a percentage of total lipids, compared to the levels of untreated cells (control). As can be seen, the enantiomer [-] produces increases in SM, which indicates that it is an activator of the enzyme EC 2.7.8.27, and the enantiomer produces a reduction in the levels of SM (especially obvious at a concentration of 100 μΜ [+ J20HOA), which indicates that it is an inhibitor of This enzyme In this context, and without affecting other levels, we have been able to verify that mixtures containing this enantiomer induce activation of the enzyme EC 2.7.8.27, the racemic mixture being a special case of molecular mixing, although a positive effect has also been proven in mixtures between the enantiomer [-] and different vehicles or different therapeutic compounds. B) Effect of forms [+] (also R enantiomer: black columns), [-] (also S enantiomer: white columns) and racemic (gray columns) on the levels of SM in U118 cell membranes (mean values after 24 h incubation with 100 μΜ). The treatments used were: vehicle (Control), 2-hydroxipalmitoleic acid (20H16: 1), 2-hydroxyoleic acid (20H18: 1); 2-hydroxylinoleic acid (20H18: 2); 2-hydroxy-ot-linolenic acid (20H18: 3a); 2-hydroxy-y-linolenic acid (20H18: 3g).

Figure 8. This figure shows the effect of racemic 20HOA (+/-) and optical isomers [-] 20HOA and [+ J20HOA on different pathological processes in animal models. A. Effect on the volume of tumors derived from human lung cancer cells (A549): immunosuppressed mice were infected with human lung cancer cells and 7 days later (when the tumors were observable) vehicle treatments were started (control ), Racemic 20HOA (20HOA), and the optical isomers [-] 20HOA and [+] 20HOA (600 mg / kg · day, 15 days, oral). The bars correspond to mean values ± SEM of the increase in volume (percentage with respect to the volume in control animals at the beginning of the treatment, considered as 100%) of the tumors (n = 6). Treatment with racemic 20HOA induced significant reductions (P <0.01) with respect to control. The optical isomer [-] 20HOA induced significant reductions compared to all other treatments (P <0.01). B. Effect on blood pressure: hypertensive rats (SHR strain) were treated with vehicle (Control), racemic 20HOA (20HOA) and their optical isomers [-] 20HOA and [+] 20HOA (600 mg / kg, every 12 h, 15 days, oral). All molecules induced significant reductions (P <0.01) in systolic blood pressure (mmHg, ordinate axis, and) of SHR rats, the [-] 20HOA isomer being the most potent in inducing hypotensive effects. Mean values ± SEM of systolic pressure are shown (n = 6). C. Effect on body weight: SHR rats were treated with vehicle (Control), racemic 20HOA (20HOA) and their optical isomers [-] 20HOA and [+] 20HOA (600 mg / kg · day, 5 days, oral). All molecules induced significant reductions (P <0.01) in the weight of the animals from the third day of treatment, with the [-] 20HOA isomer being the most potent in inducing body weight reduction. Mean values ± SEM of body weight (ordinate axis) versus treatment time in days (abcissa axis, n = 6) are shown.

D. Effect on cholesterol and triglyceride levels: SHR rats were treated with vehicle (Control), racemic 20HOA (20HOA) and their optical isomers [-] 20HOA and [+ J20HOA (600 mg / kg, 12h, 15 days, oral ). All molecules induced significant reductions (P <0.01) in the levels of triglycerides (TG) and total cholesterol (CHOt), the optical isomer [+ J20HOA being the most potent in inducing reductions in the levels of these lipids. Mean values ± SEM of serum lipid levels expressed in mg / dl (ordinate axis; n = 6) are shown. E. Effect on glycemia (plasma glucose levels): SHR rats were treated with vehicle (Control), racemic 20HOA (20HOA) and their optical isomers [-] 20HOA and [+ J20HOA (600 mg / kg, 12h, 15 days) , oral). The racemic compound and the [- J20HOA isomer produced significant reductions (* P <0.05; ** P <0.01) in plasma glucose levels (mg / dL, ordinate axis) The enantiomer [+] produced a non-significant reduction in these conditions. Mean values ± SEM of serum lipid levels expressed in mg / dl (ordinate axis; n = 6) are shown.

Figure 9. Regulation of enzyme activity 2.3.1.50. The ordinate axis (y) shows the levels of incorporation of [ ³ H] palmitate (dpm / mg protein) in different lipid fractions of U118 cells incubated in the absence (Control, white bars) or presence of 20HOA (black bars). Only a significant increase in radioactivity was found in the sphingomyelin (SM) and ceramide (Cer) fractions, which clearly indicates the selective activation of EC 2.7.8.27 and the enzyme 2.3.1.50.

Figure 10. Regulation of enzyme activity 1.14.19.1. The ordinate (Y) axis shows the levels of incorporation of [ ³ H] oleate (dpm / mg protein) into membranes of Ul 18 cells incubated in the absence (Control, white bar) or presence of 20HOA (black bar). The reduction in incorporation of tritiated oleic into the lipids of cell membranes incubated in the presence of 20HOA demonstrates the inhibition of the enzyme 1.14.19.1.

DETAILED DESCRIPTION OF THE INVENTION The following are examples that illustrate the detailed and preferred embodiments of the invention, without limiting the scope of protection thereof.

Example 1. Methodology for obtaining racemic compounds and separating the enantiomers [+] and [-] of the 2-hydroxy derivatives of fatty acids.

PROCEDURE Synthesis of the racemic compound.

 The methodology described below details the synthesis of sodium salts of 2-hydroxy fatty acids with a purity of 99%, by generating the oleic acid dianion by reaction with LDA and subsequent hydroxylation with molecular oxygen; The crude obtained is treated with NaOH to form the sodium salt and purified by two recrystallizations in MeOH / water (avoiding the use of chromatographic columns).

With this methodology can be obtained from grams of the final product to tons of it. The purity of the product can have pharmaceutical quality and be carried out under "GMP" (Good Manufacturing Practice) conditions, which is indicated for human consumption.

Scheme

 The reaction scheme in two steps, hydroxylation and purification, is shown below.

iv) HCI Where R is: - (CH ₂ ) _fl - (CH = CH-CH ₂ ) é- (CH ₂ ) _c -CH ₃ , provided that the sum of total carbons of R is equal to or greater than 12 or equal to or less than 18 (for total lengths of molecules between 14 and 20 C).

Process description

Step 1: Hydroxylation

In a 100 L inertized enameled reactor, a solution of diisopropylamine (1.7 Kg) in THF (11 L) is cooled below 5 ° C. «-BuLi (23% in hexanes) (4.6 Kg) is added to the solution in portions without the temperature exceeding 17 ° C. At the end of the addition, an equivalent of DMPU (1.0 Kg) and a solution of oleic acid (2.0 Kg) in THF (2.6 L) are added, without the temperature exceeding 20-25 ° C. The reaction mixture is heated at 50 ° C for 30 min and then cooled again to 20 ° C. At this point a bullet of 0 ₂ is connected and bubbled for about 45 min with a flow rate of 25 L / min. After the bubbling, the solution is cooled to 10 ° C and hydrolyzed with 3 M HC1 (20 L) to pH 1, ensuring that the temperature does not rise above 40 ° C. The crude product is extracted in ethyl acetate (6 L), washed with a mixture of 3 M HC1 / brine (1: 1) and sodium bisulfite (9 L) and brine (until leaving the pH at an equal value or less than 3) and the solution is concentrated to dryness in rotary evaporator. Approximately 2 kg of crude oil is obtained with a purity> 70%. · Step 2: Purification

The crude product is dissolved in 7.2 L of methanol at 40 ° C in a 10 L reactor and treated with an aqueous solution of sodium hydroxide (134 g in 1.4 L of water), increasing the T to 50 ° C for obtain a homogeneous solution. Cool to 5 ° C for at least 6 h to precipitate the sodium salt, which is filtered and washed with acetone. The solid is recrystallized twice in (10%) H ₂ 0 / MeOH (0.5 LH ₂ O / 5.0 L MeOH) at 50 ° C until complete dissolution and cooling for a minimum of 6 h at 0-5 ° C. The final product is filtered, washed with acetone and dried in vacuo. It is suspended in methanol and salt formation is completed by adding sodium methoxide. The product is completely precipitated with acetone (5 L) cooling at 0-5 ° C for at least 10 h. The solid is filtered, washed with acetone and dried in vacuo. Production of enantiomers [+] (R) and [-] (S).

 In a 1 L reactor, 100 g of the sodium salt of hydroxy derivative of fatty acid are introduced and 500 mL of sulfuric acid in ethanol (10% by weight) are added. The mixture is refluxed for 16 h and it is verified by TLC that the conversion is complete. At the end of the reaction, it is neutralized with saturated sodium bicarbonate and concentrated in vacuo to remove ethanol. 250 mL of AcOEt (ethyl acetate) are added to the aqueous emulsion to extract ethyl 2-hydroxyoleate (in case the compound used is 2-hydroxyoleic acid), dried over sulfate and concentrated to dryness.

In a 1 L reactor with mechanical agitation, 100 g of ethyl 2-hydroxyoleate (racemic mixture) are dissolved in 50 mL of MTBE. 267 mL of phosphate buffer (1 M, pH 7) and 1.3 g of pseudomonas-derived AK lipase are added to the solution. The emulsion is stirred at room temperature (25 ° C) until 50% conversion (HPLC, Luna C8, 5μιη, MeOH / water / HCOOH) is reached. The reaction is stopped by hydrolysis with 3N HC1 until acidic pH (less than 2) is reached, extracted with MTBE and washed with brine until the water pH is greater than or equal to 5. The crude is purified by crystallization to separate the fraction of hydrolyzed acid (60% enantiomeric excess) and that of the unhydrolyzed ester (75% enantiomeric excess).

Each fraction requires two reprocesses, repeating the esterification / hydrolysis procedure separately, until reaching an ee> 95% for each enantiomer, which is hydrolyzed and transformed into the final sodium salt. The final yield of the process is 40-50% (20-25% of each enantiomer).

Methodology used

The conditions described for kinetic resolution are the best obtained in the optimization process, after studying two lipases (one derived from Pseudomonas and another from Candida Antarctica), solvents (aqueous, organic or biphasic), temperatures, ionic strength, agitation, pH and dilution. The most appropriate way to follow the hydrolysis reaction and the purification of the products has also been determined. Therefore, the method for obtaining enantiomers of hydroxylated fatty acid derivatives is different from any of those that can be found in the literature, most of which require high dilutions and purifications by chromatography. Process scaling

 The method has been carried out at scales of 1-150 g, obtaining similar and reproducible results. The enantiomers can be separated by crystallization, which facilitates the kilos scale process. The loss of performance is due to the manipulation and workup processes, however, new impurities are not generated and the washing and crystallization waters could be combined and reprocessed again.

Example 2. Tumor cells have lower levels of SM in their membranes, which increase after treatment with the molecules of the invention.

The levels of MS, cultured in the absence (control) or presence of 200 μΜ of [-] 20HOA, were measured in normal cell membranes (MRC-5 and IMR90) and tumor cells of various kinds (see Table 2). In all cases, it was detected that the tumor cell membranes contain levels of MS around half or a third of the levels presented by normal cells (Figure 1). Therefore, it can be ensured that the concentration of SM is a biomarker that indicates the tumorigenicity of human cells. On the other hand, treatments with the molecules of the present invention induce an increase in SM in tumor cells that does not occur naturally. In this way, the detection of SM by lysenin and subsequent coupling of a fluorescent antibody and detection by confocal fluorescence microscopy, shows a greater marking (i.e., a greater amount of SM) in the tumors of animals treated with the molecules of the present invention (Figure le). Similarly, other detection methods based on lysenin binding to membrane MS and detection by spectroscopic tests in solution or by ELISA show how tumor cell membranes exhibit a higher level of SM only when incubated in the presence of molecules of the present invention (Figure 1). For example, determination by fluorescence spectrophotometry in solution of the levels of SM is a very simple, fast and efficient type of analysis (Figure ID). Thus, in the present invention a diagnostic method has been generated for the detection of the described pathological processes and pathologies in which the levels of MS are altered which allows to predict whether the treatment with the molecules of the present invention can be effective, thus as for monitoring the efficacy of the therapy applied using the molecules of the present invention and others of similar activity. From this knowledge, the present invention also It includes diagnostic kits to assess a priori the potential utility of this therapy and to track the efficacy of treatment with the molecules of the present invention of these diseases a posteriori.

Table 2. Effect of [-] 20HOA on sphingomyelin levels.

(1) Antiproliferative effect: + cell growth inhibition, - absence of effect. (2) Technique used for the analysis: 1) TLC or HTPLC, 2) gas chromatography, 3) image analysis, 4) fluorescence spectroscopy, 5) confocal or fluorescence microscope.

Example 3. 20HOA raises the levels of MS by activating the enzyme EC 2.7.8.27.

20HOA raises the levels of SM in treated tumor cells as indicated above. The increases in SM in tumor cells treated with 20HOA were dependent on the treatment time and the concentration used in them (Figure 2), which demonstrates the specificity of this molecule. SM is a membrane lipid that prevents the binding of certain molecules involved in cell proliferation, such as the Ras protein. In this sense, Ras levels were measured by confocal microscopy, using a fluorescently labeled antibody, and it could be detected that the tide passed from the membrane (Figure 6: 95-99% of the total fluorescence detected in membranes before treatment ) to the cytoplasm (94-97% of the fluorescence detected inside the cell after treatment with 20HOA) in human glioma, lung cancer and leukemia cells. The translocation of Ras from the membrane to the cytoplasm implies the absence of productive interactions between Ras and the Receptor-Tyrosine-Kinase (RTK) or between Ras and Raf, so that the proteins of the MAP kinase pathway do not receive activation and Tumor cells stop proliferating and cell death programs begin. The changes induced by the molecules of the present invention in cell physiology have as intermediate effects (prior to the death of cancer cells or the regulation of activity in other cells) the induction of dramatic reductions in DHFR levels and dramatic increase of PFAG levels (Figure 6). Another effect associated with the reduction of cell proliferation is the increase in nuclear MS levels. The 20HOA treatments resulted in increases in nuclear MS levels (Figure 3), demonstrating that cell proliferation inhibition is induced. Example 4. Regulation of the enzyme EC 2.7.8.27 depends on the molecular structure of the fatty acid.

In vitro studies, using the specific fluorescent substrate of the enzyme EC 2.7.8.27, BD-Cer, indicate that 20HOA interacts directly with it, the enzymatic activation being seen from the first minutes of incubation in cell cultures (Figure 4A). The rapidity of this phenomenon clearly indicates the direct and effective interaction between the 20HOA enantiomer and the enzyme EC 2.7.8.27. Therefore, the present invention refers to the use of the aforementioned compounds as activators (enantiomer [-]), or inhibitors (enantiomer [+]) specific to the enzyme EC 2.7.8.27.

On the other hand, the activation of the enzyme EC 2.7.8.27 depends on the number of carbon atoms that the fatty acid has, so that fatty acids with more than 20 C atoms do not produce significant changes in the activity of this enzyme ( Figure 5). In addition, other requirements for activation of the enzyme EC 2.7.8.27 are the presence of an OH group on carbon 2 and one or more double bonds, since the presence of other radicals (such as H or CH ₃ ) and the lack of Double bonds in the fatty acid structure gave rise to inactive molecules (Figure 5). MS has a molecular structure that determines the effects produced on cell physiology and justifies the reversal of different pathological processes. Its bulky polar head prevents the anchoring of Ras to the membrane through its isoprenyl moiety (which prefers membrane regions with small polar head phospholipids, such as phosphatidylethanolamine), which in turn prevents subsequent signaling through the proteins of the MAP kinase chain. The inactivation of this pathway induces, among other phenomena, the inhibition of tumor growth (Figure 8A and Table 2). The compounds of the present invention have been shown to be able to regulate positively (increases) or negatively (decreases) the levels of SM in the cells. Since the membrane composition is critical for its proper functioning, both the activators (S or - enantiomers of the enzyme EC 2.7.8.27, and its inhibitors (R or + enantiomers) have therapeutic activity, as It has been shown here. This activation is based on structural principles and follows the structure-function parameters of any other molecule with therapeutic activity. Example 5. The [-] isomers of C18 fatty acids, such as [-] 20HOA, are specific activators of the enzyme EC 2.7.8.27.

The relative structural conformation of the hydroxyl groups of carbons 1 and 2 (Cl and C2) gives rise to two enantiomers or optical isomers that are called [-] (corresponding to the structural conformation S) and [+ (corresponding to the structural conformation R)]. Through the process described in the present invention (Example 1), the racemic compound has been synthesized and isolated, whose activity to regulate both the EC 2.7.8.27 enzyme positively and negatively has become apparent, and the enantiomers or optical isomers, and has measured their ability to activate or inhibit the enzyme EC 2.7.8.27. Figure 7A shows the activation of said enzyme, measured through the levels of SM in U118 human glioma cell membranes, after 24 hours of incubation in the presence of 50 and 100 mM of each of the isomers, as well as the racemic mixture (which contains approximately the same amount of each of them). Figure 7A shows how racemic 20HOA produces significant increases in levels of SM in U118 cell membranes. Likewise, the optical [-] 20HOA isomer (corresponding to the S enantiomer) is capable of producing even greater increases in the levels of SM. Therefore, and in the specific case of this enzyme, the presence of mixtures with a certain percentage of [- J20HOA (the racemic compound being a special case) behaves as a specific activator of EC 2.7.8.27. On the contrary, the optical isomer [+ J20HOA not only does not induce increases in SM, but also produces a reduction in the levels of SM in membranes. These data indicate that the optical isomer [-] 20HOA is a specific activator of the enzyme EC 2.7.8.27 and that the optical isomer [+ J20HOA (ie, the R enantiomer) is a specific inhibitor of this enzyme. Therefore, the present invention protects the use for therapeutic applications of the optical isomer [-] 20HOA (S) as activator of the enzyme EC 2.7.8.27 and of the optical isomer [+ J20HOA (R) as a specific inhibitor of this enzyme. These results can be extended to all unsaturated fatty acids between 14 carbon atoms or more and 20 carbon atoms or less, which have one or more double bonds and a hydroxyl radical on the C2 carbon (alpha carbon: Figure 7B). Example 6. The [-] and [+] isomers of C18 fatty acids, such as [-] 20HOA and [+ J20HOA, used as therapeutic agents for the treatment of human diseases.

The enantiomer [-] 20HOA and derivatives shown in this invention have therapeutic applications in different areas, such as cancer treatment, obesity, cardiovascular pathologies, diabetes, metabolic syndrome, spinal cord injury, Alzheimer's disease and other processes. Figures 6 and 8; Tables 2-5). On the other hand, the enantiomer [+ J20HOA had a positive effect on cholesterol and triglyceride levels, since it induced significant reductions in the plasma levels of these lipids, whose elevated levels are considered negative for human health (Figure 8 ). Therefore, the therapeutic effects of each of the isomers, as well as the racemic mixture, in different pathological models were studied.

Figure 8A shows the effect of oral treatments (15 days, 600 mg / kg daily, n = 6) with racemic 20HOA, and enantiomers [-] 20HOA and [+ J20HOA, on the volume of cancer-derived tumors of human lung (A549 cells) in nude mice. As can be seen, the effect of the optical isomer [-] 20HOA is superior to that of the racemic. On the other hand, after 15-day treatments with the optical isomer [+ J20HOA there were no significant changes in the volume of the tumors. This result demonstrates that the [-] 20HOA isomer is what activates the enzyme EC 2.7.8.27 and has the antitumor therapeutic activity. On the other hand, the antitumor activity of these compounds in different human cancer lines was investigated (see Tables 2, 4 and 5). In this sense, all the [-] enantiomers of the present invention demonstrated greater potency than the racemic and the [+] enantiomer for the treatment of cancer, which corresponds to significantly lower values in the IC50 to inhibit the growth of cancer of human lung (line A549). Moreover, the activity of these enantiomers was evidenced against a wide variety of human cancers of different kinds, so it can be ensured that the enantiomers [-] of 2-hydroxylated fatty acids have a high potency for the treatment of any type Of cancer. This potency turned out to be somewhat higher for salts (mainly the sodium salt of these molecules) than for free fatty acid (results not shown). In any case, the enantiomers [-] both in acidic form and in salt, always they presented greater potency than any of the alternative molecular forms to inhibit tumor growth.

In addition, the effect of the above-mentioned isomers on blood pressure, body weight, triglyceride and cholesterol levels and blood glucose (plasma glucose) in SHR rats (Figure 8B, 8C, 8D and 8E) was studied. Similarly to what was indicated above, there was a greater therapeutic effect when the [- J20HOA isomer was used in the control of blood pressure, body weight and glycemia, while the [+ J20HOA enantiomer had a greater therapeutic effect for the treatment of hypercholesterolemia and hypertriglyceridemia. Thus, the animals treated with the [- J20HOA isomer showed reductions in blood pressure greater than 60 mm Hg their systolic pressure, while those treated with the [+ J20HOA isomer and with the racemic product had a remarkable therapeutic effect but of less amplitude . Similarly, between days 1 and 5 of treatment, rats treated with the racemic lost 11 grams of weight (approximately 3% of body weight), while animals in the group treated with the enantiomer [-] 20HOA lost 21 grams and the animals of the group treated with the enantiomer [+ J20HOA only lost 7 grams of weight. On the other hand, a superior effect of the [+ J20HOA enantiomer in reducing triglyceride (TG) and total cholesterol (CHOt) levels was observed (Figure 8D). In this sense, the baseline plasma levels of TG in SHR rats are 108 mg / dl, and were reduced to 56 mg / dl after 2-week treatments with enantiomer [+] 20HOA. On the other hand, treatment with the [+ J20HOA enantiomer induced a reduction in plasma TG levels up to 47 mg / dl, while the [-] 20HOA enantiomer only produced a modest decrease to 81 mg / dl. In parallel, CHOt levels were reduced from 73 mg / dl to 43 mg / dl (racemic), 36 mg / dl ([+ J20HOA enantiomer) and 59 mg / ml ([-] 20HOA enantiomer), respectively. Finally, treatment with the enantiomers of the present invention also produced significant reductions in plasma glucose levels (Figure 8E). In this case, both the racemic product (20HOA) and the enantiomer [-] 20HOA induced significant drops in glycemia in rats (P <0.05 and P <0.01, respectively: Figure 8E). However, the R ([+] 20HOA) enantiomer induced a modest decrease that did not reach statistical significance. Therefore, this invention shows that the optical isomers [-] [+] of unsaturated fatty hydroxy acids of 18 C atoms are effective molecules with therapeutic activity for the treatment of the pathologies indicated above. In all the cases studied, one and only one of the enantiomers showed to have an activity superior to that of the other molecular forms (statistical significance always P <0.05). In this way, the [-] enantiomer was shown to be more effective in curing cancer, obesity, diabetes, hypertension, etc., while the enantiomer [+] showed to be more effective in controlling hypercholesterolemia or hypertriglyceridemia. In these cases, the racemic compound was shown as an intermediate situation, capable of emulating the positive effects of any of the optical isomers, but with a lower potency, due to the lower concentration of the active enantiomer in each case. In addition, the use of the unsuitable enantiomer induced unwanted side effects that were avoided at therapeutic doses with the most active enantiomer (Table 3).

Example 7. Efficacy, toxicity and side effects of the enantiomers of the invention.

This example reflects the study conducted in immunosuppressed mice infected with human glioma cells (SF767) and treated for 15 days (oral) with the indicated doses (Table 3). This table shows the volume of the tumors at the end of the treatment and the symptoms observed during the days that it lasted. Note, once again, that the efficacy of the [-] 20HOA enantiomer is superior to that of the 20HOA racemic compound, and that the [+ J20HOA enantiomer showed no activity after 15 days of treatment at the indicated doses.

On the other hand, the dose that induced reductions of approximately one third of the tumor volume did not induce any side effects in animals treated with [- J20HOA (50 mg / kg), while animals treated with the racemic compound 20HOA at Doses that induced similar reductions in tumor volume (600 mg / kg) showed significant side effects (Table 3). In addition, at 200 mg / kg, the [-] 20HOA enantiomer induced tumor volume reductions of 89% without observable adverse effects, while at the same dose, the racemic compound only induced or 23% reductions in the volume of the tumors and some of the animals presented adverse effects in response to treatment. From these results it is deduced that the [-] 20HOA enantiomer has a greater potency and that at the maximum therapeutic doses it does not produce adverse effects, while the racemic compound has a more modest effect and induces certain unwanted effects at therapeutic doses. In the treatment of tumor processes, the differences in efficacy between the [-] 20HOA and the racemic enantiomer can mean differences of months or years in the life expectancy of patients. Moreover, differences in efficacy can result in the cure or not of a certain cancer in a patient. On the other hand, differences in toxicity at therapeutic doses can result in a higher quality of life in patients receiving the enantiomeric form [-] 20HOA. Since the mechanism of action of the compound is related to the activity on the enzyme EC 2.7.8.27 and that the enantiomers [-] and [+] have a contrasted effect on said enzyme (Figure 7), the administration of the enantiomer ([- ] 20HOA) is much more effective than that of the racemic compound, reversing part of the therapeutic effect of the latter. Table 3

 1. Variation of tumor size after 15 days of treatment (oral) at the indicated dose with respect to untreated animals (n = 6 in all groups). In this case, the 100% value was assigned to the tumor volume of the control animals (treated with vehicle) at the end of the 15-day treatment.

2. Registered symptoms and percentage of animals in which it has been observed. Behavioral symptoms include reduced mobility for at least 30 minutes, ruffled hair, jumping (jumping), or confinement in a corner of the cage. Studies conducted with the racemic compounds and with the enantiomers of the present invention demonstrate that in all cases the enantiomer [-] has a higher antitumor potency than the other molecular forms to inhibit the growth of human cancer cells (A549, Table 4 ). In this sense, the concentrations that inhibit the growth of tumor cells (IC ₅₀ ) are lower for the S ([-]) enantiomer in all the tumor lines studied (Table 5). On the contrary, these compounds did not induce the death of normal cells (MRC-5, IMR90). In all cases, the molecules used were sodium salt. Table 4. Effect of the compounds of the invention on the growth of human lung cancer cells, A549

Ι (Ι ₅ ο (μΜ)

 Racemic [-] [+]

 201-116: 1 192.7 ± 9.3 110.2 ± 14.0 * 299.0 ± 34.2 *

201-118: 1 94.Ü8.8 52.6 ± 9.5 ** 133.3 ± 9.8 *

20H18: 2 105.0 ± 4.1 69.9 ± 11.2 * 186.3 ± 21.9 *

20H18: 3rd 219.2 ± 15.4 146.1 ± 10.9 * 351.4 ± 22.7 **

20H18: 3g 206.5 ± 21.7 155 ± 7.5 * 341.8 ± 31.5 **

* P <0.05; ** P <0.01: significantly different from the racemic; P <0.001 with respect to the enantiomer [+]. Example 8. Regulation of the compounds of the invention on the activity of the enzyme EC 2.3.1.50.

U118 human glioma cells were incubated in the presence or absence (control) of 20HOA (200 μΜ, 24 h) and then incubated with [ ³ H] palmitate for 5 minutes. After said incubation periods, cell lipids were extracted and separated by TLC. The bands corresponding to each lipid species were extracted and the amount of radioactive palmitate incorporated was measured by liquid scintillation. As can be seen, there was not only an important incorporation in the SM fraction (indicator of activation of EC 2.7.8.27), but also in the ceramide fraction, which demonstrates the activation of the enzyme EC 2.3.1.50 ( serine palmitoyl transferase). Therefore, it can be concluded that 20HOA is a specific activator of the enzyme EC 2.3.1.50 (Figure 9). Table 5. Effect of racemic 20HOA and its enantiomers on the growth of several lines of different types of human cancer. IC ₅₀ values (μΜ).

Cell Line Cancer Type 20HOA Racemic H20HOA [+] 20HOA

 U118 Glioma (human) 142.3 97.8 211.6

SF767 Glioma (human) 214.8 102.1 304.1

U87 Glioma (human) 81.6 53.5 148.3

SH-SY5Y Neuroblastoma 119.1 64.4 193.4

 (human)

 A549 Lung cancer 94.1 52.6 133.3

 (human)

 Jurkat Leukemia (human) 62.9 35.7 97.2

U937 Lymphoma (human) 183.2 136.3 213.5

HepG2 Liver cancer 41.4 32.9 76.7

 (human)

 MDA-MB-231 Breast cancer 243.7 145.2 328.0

 (human)

 PC3 Prostate Cancer 137.5 87.0 232.8

 (human)

 BXPC3 Pancreatic Cancer 98.3 61.8 154.1

 (human)

 HeLa Uterine Cancer 126.8 74.5 205.7

 (human)

 HT29 Colon cancer 224.6 159.4 341.6

 (human)

 A375 Malignant melanoma 272.9 163.1 407.3

 (human)

 IMR90 Fibroblast no> 5,000> 5,000> 5,000

 tumor (human)

 MRC-5 Fibroblast no> 5,000> 5,000> 5,000

 tumor (human) Example 9. Regulation of the compounds of the invention on the activity of the enzyme EC 1.14.19.1.

Oleic acid is synthesized from stearic acid through the activity of the enzyme stearoyl-CoA desaturase (EC 1.14.19.1). This enzyme is crucial in lipid metabolism, since it is limiting in the synthesis of fatty acids. To study whether this reduction was due to EC regulation 1.14.19.1, Ul 18 cells were incubated in the presence or absence of 20HOA (200 μΜ, 24 h) and, subsequently, in the presence of [ ³ H] oleate (5 minutes ). It was found that the incorporation of radioactive oleic acid into U118 cell membranes incubated in the presence of 20HOA was significantly lower than in control cells (Figure 10). This result indicates that 20HOA is a potent inhibitor of CD 1.14.19.1, whose regulation has been suggested to be important in the treatment of different human pathologies.

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Claims

1. Enantiomer [-] or [+] of a compound of Formula I:

HOOC-HOCH- (CH2) „- (CH = CH-CH2) _é - (CH2) _c -CH ₃

 (I)

or its pharmaceutically acceptable salts, characterized in that a, b and c can take independent values between O and or, with the proviso that the total number of carbons is <20.

2. Enantiomer according to claim 1, resulting from the selection of at least one of the following combinations of values a, b and c:

 a = 6, 6 = 1 and c = 4

 a = 6, 6 = 1 and c = 6

 a = 6, b = 2 and c = 3

 a = 6, b = 3 and c = 0

 a = 3, b = 3 and c = 3

3. Enantiomer according to claim 2, characterized by the formula:

[-] HOOC-HOCH- (CH ₂ ) _é - (CH = CH-CH ₂ ) i- (CH ₂ ) _(í -CH ₃ or its pharmaceutically acceptable salts.

4. Enantiomer according to claim 2, characterized by the formula:

[+] HOOC-HOCH- (CH ₂ ) _é - (CH = CH-CH ₂ ) i- (CH ₂ ) _(í -CH ₃ or its pharmaceutically acceptable salts.

5. Enantiomer according to claims 1 to 4, characterized in that the pharmaceutically acceptable salt is a sodium salt.

6. Pharmaceutical composition comprising at least one enantiomer of claims 1 to 5, and / or at least one of its pharmaceutically acceptable salts, and, optionally, any pharmaceutically acceptable carrier.

7. Use of at least one enantiomer of claims 1 to 5, or at least one of its pharmaceutically acceptable salts, for the preparation of a pharmaceutical composition for the treatment and / or prevention of pathologies whose common etiology is structural alteration and / or functional membrane cell, due to deregulation of the activity of the enzyme EC 2.7.8.27, the level of sphingomyelin, the level of PFAG or the level of DHFR.

8. Use of the enantiomer of the formula [-] HOOC-HOCH- (CH2) ¿- (CH = CH-CH2) ₇ - (CH2) <5-CH _3; and / or at least one of its pharmaceutically acceptable salts, according to claim 7, for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of pathologies whose etiology is the structural and / or functional alteration in the cell membrane due to a EC 2.7.8.27 enzyme activity deficit, at an abnormally low level of sphingomyelin, at an abnormally low level of PFAG or at an abnormally high level of DHFR.

9. Use according to claim 8, characterized in that the pathology is selected from: cancer, preferably liver cancer, melanoma, prostate cancer, breast cancer, pancreatic cancer, leukemia, uterine cancer, colon cancer, cancer of brain or lung cancer

10. Use according to claim 8, characterized in that the pathology is selected from: vascular pathologies, preferably hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia; or metabolic pathologies, preferably diabetes, metabolic syndrome or obesity.

11. Use of the enantiomer of formula [+] HOOC-HOCH- (CH2) ¿- (CH = CH-CH2) ₇ - (CH2) <5-CH _3; and / or at least one of its pharmaceutically acceptable salts, according to claim 7, for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of pathologies whose etiology is the structural and / or functional alteration in the cell membrane due to a excess in the activity of the enzyme EC 2.7.8.27, at an abnormally high level of sphingomyelin, at a normally high level of PFAG or at an abnormally low level of DFIFR.

12. Use according to claim 11, characterized in that the pathologies are preferably hypercholesterolemia, hypertriglyceridemia and cystic fibrosis.

13. In vitro method for the selection of candidate compounds useful in the treatment and / or prevention of pathologies whose etiology is the structural and / or functional alteration of lipids located in the cell membrane, which includes the evaluation of the enzyme activity EC 2.7.8.27, of the level of sphingomyelin, of the level of PFAG or of the level of DHFR in the presence of said candidate compound.

14. In vitro method for the prognosis / diagnosis of pathologies whose etiology is the structural and / or functional alteration of lipids located in the cell membrane, which includes the determination of the deregulation of the activity of the enzyme EC 2.7.8.27, of sphingomyelin level, PFAG level or DHFR level

15. In vitro method for the prognosis / diagnosis of pathologies whose etiology is the structural and / or functional alteration of the lipids located in the cell membrane, according to claim 14, which comprises the determination of the deficit in the activity of the enzyme EC 2.7 .8.27, the presence of an abnormally low level of sphingomyelin, an abnormally low level of PFAG or an abnormally high level of DFIFR.

16. In vitro method according to claim 15, wherein the pathologies are selected from: cancer, preferably liver cancer, melanoma, prostate cancer, breast cancer, pancreatic cancer, leukemia, uterine cancer, colon cancer, cancer of brain or lung cancer.

17. In vitro method according to claim 15, wherein the pathologies are selected from: vascular pathologies, preferably hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia; or metabolic pathologies, preferably diabetes, metabolic syndrome or obesity.

18. In vitro method for the prognosis / diagnosis of pathologies whose etiology is the structural and / or functional alteration of the lipids located in the cell membrane, according to claim 14, which comprises the determination of the excess in the activity of the enzyme EC 2.7 .8.27, of the presence of a level abnormally high sphingomyelin, an abnormally high level of PFAG or an abnormally low level of DHFR

19. In vitro method according to claim 18, wherein the pathology is hypercholesterolemia, hypertriglyceridemia or cystic fibrosis.

20. Kit for use in the prone / diagnostic method of claims 14 to 19 comprising useful means for determining the activity of the EC enzyme 2.7.8.27, the level of sphingomyelin, the level of PFAG or the DHFR level

21. Kit according to claim 20, comprising useful means for the direct determination of sphingomyelin, its precursors, its derivatives or for the indirect measurement of sphingomyelin.

22. Kit according to claim 21, wherein the precursor is ceramide, the derivative is BD-Cer or BD-SM and indirect measurement is made through lysenin.

23. Kit according to claims 20 to 22, wherein the useful means comprise: TLC and FtPTLC technique, gas chromatography, image analysis, absorption or fluorescence spectroscopy, fluorescence or phase contrast optical microscopy, microscopy confocal, immunoblot, polymerase chain reaction, ELISA, RIA, EIA, dot blot or any other related technique.

24. Method of synthesis of 2-hydroxy derivatives of fatty acids and subsequent isolation and purification of their enantiomers comprising:

 a) Hydroxylation of fatty acid in acidic medium and subsequent purification of the sodium salt, sodium 2-hydroxyoleate in the case of starting from oleic acid.

b) Esterification of the racemic mixture of sodium 2-hydroxyoleate, or any of the hydroxylated fatty acids of the present invention, with a solution of sulfuric acid in ethanol, preferably 10%. c) Selective enantium hydrolysis of the ester, catalyzed by a lipase, preferably pseudomone lipase and preferably at 25 ° C. d) Control of the progress of the reaction preferably by liquid chromatography.

 e) Hydrolysis of the mixture with diluted HC1 to pH less than 2. f) Extraction of the products and washing of the organic phase.

 g) Removal of the solvent in vacuo to obtain a crude with the mixture of acid and ester.

 h) Separation of acid and ester preferably by crystallization of the first.

 i) Return to step a) in the case of acid and step b) in the case of the ester to start reprocessing each of the isolated fractions (acid and ester) until the desired enantiomeric purity is obtained.

25. Use of a compound of Formula I

HOOC-HOCH- (CH2) „- (CH = CH-CH2) _é - (CH2) _c -CH ₃

 and / or at least one of its pharmaceutically acceptable salts for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of pathologies whose common etiology is the structural and / or functional alteration of the cell membrane, due to the deregulation of activity EC 2.7.8.27 enzyme, sphingomyelin level, PFAG level or DHFR level.

26. Use of a compound of Formula I

HOOC-HOCH- (CH2) _fl - (CH = CH-CH2) _/ , - (CH2) _c -CH ₃

 (I)

as activator of the enzyme EC 2.7.8.27, and / or as activator of the enzyme EC 2.3.1.50, and / or as inhibitor of the enzyme EC 1.14.19.1, in vitro.

27. Use according to claim 26, characterized in that the compound of Formula I results from the selection of at least one of the following combinations of values a, b and c:

 a = 6, 6 = 1 and c = 4

 a = 6, 6 = 1 and c = 6

 a = 6, b = 2 and c = 3

 a = 6, b = 3 and c = 0

 a = 3, b = 3 and c = 3